Amplified 16S rDNA Research Articles

Machine Learning Study in Caries Markers in Oral Microbiota from Monozygotic Twin Children.

In recent years, the etiology of caries has evolved from a simplistic infectious perspective based on Streptococcus mutans and/or Lactobacillus activity, to a multifactorial disease involving a complex oral microbiota, the human genetic background and the environment. The aim of this work was to identify bacterial markers associated with early caries using massive 16S rDNA. To minimize the other factors, the composition of the oral microbiota of twins in which only one of them had caries was compared with their healthy sibling. Twenty-one monozygotic twin pairs without a previous diagnosis of caries were recruited in the context of their orthodontic treatment and divided into two categories: (1) caries group in which only one of the twins had caries; and (2) control group in which neither of the twins had caries. Each participant contributed a single oral lavage sample in which the bacterial composition was determined by 16S rDNA amplification and further high-throughput sequencing. Data analysis included statistical comparison of alpha and beta diversity, as well as differential taxa abundance between groups. Our results show that twins of the control group have a closer bacterial composition than those from the caries group. However, statistical differences were not detected and we were unable to find any particular bacterial marker by 16S rDNA high-throughput sequencing that could be useful for prevention strategies. Although these results should be validated in a larger population, including children from other places or ethnicities, we conclude that the occurrence of caries is not related to the increase of any particular bacterial population.

Analysis of gut microbiome in patients with lung adenocarcinoma and lung squamous cell carcinoma

To investigate the diversity and composition of gut microbiota in patients with lung adenocarcinoma and lung squamous cell carcinoma. A single-center and case-control study was conducted to consecutively enroll a total of 27 lung cancer patients, including 15 males and 12 females, who were seen at the Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine between September 2018 to October 2020. A total of 20 cases of healthy healthy physical examiners, including 9 males and 11 females were recruited as healthy control group (HC) during the same period. Clinical data and stool samples were collected from each participants, and lung cancer patients were divided into lung adenocarcinoma group (AC, 19 patients, 8 males and 11 females) and lung squamous cell carcinoma group (SCC, 8 patients, 7 males and 1 females) according to the pathology type. Genomic DNA were extracted to amplify 16S rDNA V3-V4 region, then the Illumina MiSeq high-throughput sequencing platform and QIIME software were used for sequencing and analyzing the structure of the gut microbiota, respectively. Analysis of variance, χ2 test, K-W test were used to analyze the differences in age, gender,α diversity, and relative abundance of microbiota among the three groups. AC, SCC, and HC were aged (58.74±9.27), (63.38±6.12), and (55.65±7.79) years old, respectively. There were no difference in gender and age among the three groups (gender and age are respectively:χ2=5.155, P=0.076;F=2.598,P=0.086). And no significant difference in alpha diversity were found among the three groups (Chao and Shannon index were respectively: F=0.616, P=0.545; F=2.484, P=0.095), while β-diversity analysis indicated significant differences in the structure of intestinal flora among AC, SCC and HC (P=0.001). LEfSe analysis showed that AC and SCC both have dominant bacterials. Megasphaera (H=7.855,P=0.020) and Erysipelatoclostridium (H=7.426,P=0.024) were enriched in patients with AC, while Enterococcus (H=8.400, P=0.015), Veillonella (H=9.957,P=0.007), and Eubacterium_eligens_group (H=10.514,P=0.005) were enriched in patients with SCC. Lung cancer patients have gut microbiota imbalance, while lung adenocarcinoma and lung squamous cell carcinoma patients have no significant difference in gut microbiota diversity, but lung adenocarcinoma and lung squamous cell carcinoma have their own unique microbiota. This imbalance of the intestinal microenvironment is of great significance for studying the occurrence and development of different pathological types of lung cancer.

Pseudomonas aeruginosa Growth Inhibitor, PAGI264: A Natural Product from a Newly Isolated Marine Bacterium, Bacillus sp. Strain REB264

Increasing concerns about the emergence of antibiotic-resistant bacteria have drawn much attention for research efforts to introduce new types of antibacterial compounds among scientists. In the present report, 24 bacterial strains isolated from the coastal areas of the Persian Gulf in Iran were investigated in order to assess their potentials to produce antibacterial metabolites. Among these, the crude extract from the isolate REB264 showed promising inhibition activity on the growth of two pathogenic bacteria, Pseudomonas aeruginosa and Staphylococcus aureus. Molecular identification via sequencing of the amplified 16S rDNA gene of the strain REB264 revealed that the bacterium belonging to the genus Bacillus and was tentatively named Bacillus sp. REB264, while the sequence was deposited in GenBank database, NCBI. Optimization of the carbon and nitrogen sources of the strain REB264 culture media determined that 743 mg/l active extract was achieved when using 1% glycerol and 0.5% yeast extract as carbon and nitrogen sources, respectively. Crude extract purification to obtain the antibacterial metabolite was performed in three steps using fractionation in organic and aqueous solvents, silica gel 60 column chromatography and LH-20 gel filtration with the yield of 80%. The purified compound exhibited inhibitory effect on P. aeruginosa growth at minimum inhibitory concentration (MIC) of 15 µg/ml, while lacking antibacterial effect on S. aureus. The P. aeruginosa growth inhibitor 264 (PAGI264) compound was further purified using reversed-phase high-performance liquid chromatography (C18-HPLC) up to 95% and the structural analysis by FTIR and H1-NMR confirmed that PAGI264 is composed of alcoholic, carbonyl and phenolic functional groups. Moreover, MS data in positive mode showed that the molecular weight of the compound is 761.74 Da. Lack of toxicity to MCF-7 cell line up to 500 µg/ml and lack of the hemolytic effect on red blood cells showed that the present natural product, PAGI264, has capability to use as a lead or drug in the future.

Screening of actinobacteria-producing amylolytic enzyme in sediment from Litopenaeus vannamei (Boone, 1931) ponds in Rembang District, Central Java, Indonesia

Abstract. Ayuningrum D, Sabdaningsih A, Jati OE. 2021. Screening of actinobacteria-producing amylolytic enzyme in sediment from Litopenaeus vannamei (Boone, 1931) ponds in Rembang District, Central Java, Indonesia. Biodiversitas 22: 1819-1828. Coastal environments are dynamic places where various activities such as aquaculture take place. Waste from intensive aquaculture contains high amounts of organic material that negatively affect the pond system as well as the surrounding environment. Bioremediation using hydrolytic enzymes from microbes has been widely used as an alternative to remove organic waste because of its lower economic cost and more efficient. Microbes such as Gram-positive actinobacteria are widely known as enzyme producers. This research aimed to isolate actinobacteria from Litopenaeus vannamei pond sediment, to purify and characterize the actinobacteria based on morphological appearance, to conduct screening of amylolytic activity, and to identify the species of actinobacteria through 16S rDNA amplification as well as to construct their phylogenetic tree. Sampling was done in Rembang District, Central Java, Indonesia, with 5 sampling stations consist of 14 sampling points. Thirty-eight pure axenic cultures of actinobacteria were successfully isolated using two different media, namely IM6 and IM8. The IM8 medium promoted the most growth of 24 isolates rather than on the IM6 with only 14 isolates. Based on morphological appearance, most isolates belonged to Streptomyces-like actinomycete bacteria as many as 30 isolates and the rest 8 isolates belonged to mycelium-forming non-Streptomyces actinomycete bacteria. The screening results of amylolytic activity showed that all of the 38 actinobacteria formed halo zone with amylolytic index between 1.31-5.29. Top five highest amylolytic index was found in isolates SA4.1 (IM6), SD1.3 (IM8), SC3.3 (IM8), SC3.3 (IM6) and SC3.2 (IM8). The isolate SC3.3 (IM8) according to BLAST homology, showed a 98% similarity with Streptomyces atacamensis C60. Further research is needed to investigate the potential of isolate SC3.3 (IM8) to reduce the BOD level in the aquatic system as a bioremediator candidate.

Multilocus sequence typing and phylogenetic analysis revealed two distinct almond witches'‐broom phytoplasma subpopulations in Iran

AbstractDuring a field survey conducted in different geographical regions of Iran, phytoplasma‐like symptoms were observed in peach, almond, wild almond and GF‐677 rootstock. Based on 16S rDNA amplification, followed by nucleotide sequence and phylogenetic analyses, almond witches'‐broom (AlmWB) phytoplasma strains were found in association with symptomatic plants. Based on the single‐nucleotide polymorphisms within 16S rDNA sequence, most of the Iranian AlmWB phytoplasma strains were distinguished from the Lebanese strains. Further molecular typing, based on sequence and phylogenetic analyses of rplV‐rpsC (ribosomal proteins) and imp (immunodominant membrane protein) genes, indicated that AlmWB phytoplasma strains can be differentiated in two subpopulations: Ir, including exclusively Iranian strains; Lb, comprising strains from Lebanon and Iran. Moreover, the selection pressure analysis of imp gene sequences suggested that these two AlmWB phytoplasma subpopulations had some distinct biological or ecological properties. Finally, results of the sequence and phylogenetic analyses performed on rplV‐rpsC and imp genes showed a considerably high genetic distance between AlmWB phytoplasma strains (16SrIX‐B and variants) and phytoplasmas belonging to other 16SrIX subgroups. The significance of genetic variation in phytoplasmas of 16SrIX group is discussed in relation to their biological characteristics and geographical distinction.

Isolation and Characterization of Rhodococcus spp. from Pistachio and Almond Rootstocks and Trees in Tunisia

The purpose of this study was to isolate and identify Rhodococcus spp. strains from almond and pistachio rootstocks and trees in Tunisia. Twenty-eight strains were identified through 16S rDNA and vicA genes amplification and sequencing. Pea bioassay was performed to determine the pathogenicity of the strains. Representative 16S rDNA and vicA sequences of eight strains from pistachio and seven strains from almond were closely related (>98% similarity) to Rhodococcus spp. accessions in GenBank. Phylogenetic analysis based on 16S rDNA sequences revealed that the yellow-colored strains clustered with phytopathogenic Rhodococcusfascians. The red and orange-colored strains were separated into a different group with R. kroppenstedtii and R. corynebacteiroides isolates. Eleven strains affected the pea seedlings’ growth and exhibited different levels of virulence. The number of shoots was significantly higher in seedlings inoculated with four Rhodococcus strains, whereas the other three strains caused up to 80% of plant height reduction and reduced root secondary growth compared to non-inoculated pea seedlings. These strains, most of which are epiphytes from asymptomatic hosts, showed strong pathogenicity during pea bioassay and were established endophytically in pea tissues. Ten att and five fas genes were detected in four strains and may represent a novel model of plant pathogenic Rhodococcus virulence. The results of our survey showed that Rhodococcus is present but not prevalent in all visited orchards of almond and pistachio rootstocks and trees. Our surveys complemented the investments being made on ornamental species in Tunisia and unveiled the presence of undocumented plant-associated Rhodococcus spp. on economically important crops.

First report of Cedecea neteri Causing Yellow rot Disease in Pleurotus pulmonarius in China.

Pleurotus pulmonarius is a popular edible fungus and widely cultivated in many areas of China. In June 2018, yellow rot (more than 10% incidence) was found on the first crop of P. pulmonarius fruiting bodies in a mushroom factory in Nanning, Guangxi Province, China. At first, yellow water-soaked lesions appeared in the infected fruiting bodies. Lesions then spread and purulent tissues were formed. Severe rot induced production of deformed fruiting bodies and offensive odor. Internal sections of the diseased tissue (approximately 0.5 × 0.5 cm) were sterilized in 75% alcohol for 30 s, rinsed three times with sterilized and deionized water, crushed and suspended in sterilized and deionized water. The suspension was spread on the Luria-Bertani (LB) medium. After incubation at 30°C for 2 days, dominant bacterial colonies were oyster white, smooth, convex, and circular. Individual colonies were transferred two times to LB medium using the conventional streak plate techniques to obtain the pure cultures. The cells were gram-negative, short rods, motile, and no capsules or endospores were observed. Using a BoJian Gram-negative bacteria biochemical analysis kit (5 CARDS, Hopebio, Qingdao, China), data were obtained and analyzed, showing that the isolated strain belongs to the Cedecea genus (positive for β-galactosidase, citric acid, arginine, sucrose, mannitol, sorbitol, D-glucose, gelatin hydrolysis and VP test but negative for H2S, urease, oxidase, indole, rhamnose, melibiose, amygdalin, lysine, ornithine, lactose, inositol and arabinose). Amplified 16S rDNA gene sequences (1,424 bp, GenBank accession No. MT925570) of the isolate using the universal primers 27f and 1492r (Lane 1991) exhibited 99.86% identity with Cedecea neteri M006 (CP009458.1). Based on its morphological characteristics, 16S rDNA sequences, and biochemical test results, the strain was identified as C. neteri. Pathogenicity tests for this strain were performed with bacterial suspensions (approximately 1 × 108 CFU/ml) after growing for 24 h in LB medium at 30°C. Mycelia of P. pulmonarius were cultivated for 60 days in plastic bags. Then young fruiting bodies were formed after induced with low temperature stimulation to serve as a host source. The prepared bacterial suspensions were directly sprayed onto the surface of three bags of fruiting bodies; another three bags were sprayed with sterilized and deionized water as negative control. All inoculated fruiting bodies were then incubated at 20°C with 90 to 95% relative humidity. All experiments were repeated three times. After 2 days, all the fruiting bodies inoculated with the bacterial suspensions showed yellow water-soaked lesions, and the normal growth of the fruiting bodies was inhibited. An offensive odor then developed along with a severe soft rot that was similar to the disease symptoms observed under natural conditions. The fruiting bodies of negative control were growing healthily with no symptoms. Koch's postulates were fulfilled by isolating bacteria from lesions on artificially inoculated fruiting bodies that were identical to the original isolates based on morphological characteristics, 16S rDNA sequences and biochemical test results. C. neteri was formally reported as a pathogen to humans that could cause bacteremia (Farmer et al. 1982). Recently, it has also been reported causing soft rot disease on mushrooms of Pholiota nameko (Yan et al. 2018) and yellow sticky disease on mushrooms of Flammulina velutipes (Yan et al. 2019). However, to the best of our knowledge, this is the first report of C. neteri-induced yellow rot disease of P. pulmonarius in China.

High throughput sequencing reveals the abundance and diversity of antibiotic-resistant bacteria in aquaculture wastewaters, Shandong, China.

The online version contains supplementary material available at 10.1007/s13205-021-02656-4.

Antifungal Activity of Bacterial Isolates from Straw Mushroom Cultivation Medium against Phytopathogenic Fungi

Several bacteria were isolated from straw mushroom (Volvariella volvacea) cultivation medium. There are three potential isolates previously characterized and has growth inhibition effect against V. volvacea. This screening result lead to the further study about the inhibition activity against phytopathogenic fungi. The aim of this research is to investigate the antifungal activity of three bacterial isolates against three phytopathogenic fungi and identification of the bacteria. The method used in this study are antifungal assay using co-culture method and disk difussion assay using the filtrate of each bacteria. The profile of antifungal compound was identified using ethyl acetate extract followed by evaporation and gas chromatography (GC-MS) analysis. Identification of each isolates was performed using 16S rDNA amplification and sequencing. Three phytopathogenic fungi i.e Cercospora lactucae (InaCC F168), Colletotrichum gloeosporides (InaCC F304) and Fusarium oxysporum f.sp. cubense (F817) were co-cultured with bacterial isolates C2.2, C3.8, and D3.3. The C3.8 isolate has highest average inhibition activity either using isolate and filtrate. The result relatively consistent against three phytopathogenic fungi. The metabolite profile of C3.8 isolate showed the Bis(2-ethylhexyl) phthalate as the main compound with 97% similarity. Bis(2-ethylhexyl) phthalate has potential effect as antibacterial and antifungal compound. According to EzBioCloud and GeneBank databases, the C2.2 isolate identified as Bacillus tequilensis, C3.8 as Bacillus siamensis and D3.3 as Bacillus subtilis subsp. Subtilis. This study also shows the potential of Bacillus siamensis C3.8 as biocontrol against phytopathogenic fungi.

Isolation of Phaeobacter sp. from Larvae of Atlantic Bonito (Sarda sarda) in a Mesocosmos Unit, and Its Use for the Rearing of European Seabass Larvae (Dicentrarchus labrax L.).

The target of this study was to use indigenous probiotic bacteria in the rearing of seabass larvae. A Phaeobacter sp. strain isolated from bonito yolk-sac larvae (Sarda sarda) and identified by amplification of 16S rDNA showed in vitro inhibition against Vibrio anguillarum. This Phaeobacter sp. strain was used in the rearing of seabass larvae (Dicentrarchus labrax L.) in a large-scale trial. The survival of seabass after 60 days of rearing and the specific growth rate at the late exponential growth phase were significantly higher in the treatment receiving probiotics (p < 0.05). Microbial community richness as determined by denaturing gradient gel electrophoresis (DGGE) showed an increase in bacterial diversity with fish development. Changes associated with the administration of probiotics were observed 11 and 18 days after hatching but were not apparent after probiotic administration stopped. In a small challenge experiment, seabass larvae from probiotic treatment showed increased survival (p < 0.05) after experimental infection with a mild pathogen (Vibrio harveyi). Overall, our results showed that the use of an indigenous probiotic strain had a beneficial impact on larval rearing in industry-like conditions.

Semi-continuous desulfurization of natural gas in photobioreactor by green sulfur-oxidizing bacterial consortia isolated from hot spring

Anoxygenic phototrophic bacteria are able to oxidize and remove hydrogen sulfide from natural sources by using inorganic substrates and light. In the present study, anoxygenic phototrophic bacteria were isolated from Vartoon hot spring (east of Isfahan Province, Iran) and used for removal of sulfide compounds from natural gas in a semi-continuous photobioreactor. Mat samples that obtained from the depth of spring were introduced into Winogradsky columns and kept in the presence of sunlight for 3–4 months until green and purple layers were composed. The layers were then transferred into mineral medium and incubated under anaerobic condition. The sequence of amplified 16S rDNA in green bacteria and an amplified sequence among the gene encoding photosynthetic reaction center in purple bacteria were used for molecular identification of bacteria. The bacterial growth based on the contents of bacteriochlorophylls and the extent of elemental sulfur production by the cells were measured after ethanol extraction. Molecular identification validated the presence of Chlorobium phaeobacteroides and Prosthecochloris vibrioformis from green sulfur bacteria in the consortium. The results indicated the most increase in bacteriochlorophylls c, d, and e content of mineral medium after bacterial growth. In addition, by releasing 62.4 mmol elemental sulfur, 79.30% increase was detected in sulfur content of the medium during 3 weeks. The consortium of sulfur-oxidizing phototrophic bacteria was potentially used for natural gas desulfurization in the semi-closed photobioreactor. This system is suggested as a replacement for chemoautotrophic systems in areas where there is sufficient access to sunlight.

Peptide Nucleic Acid (PNA) Clamps to Reduce Co-amplification of Plant DNA During PCR Amplification of 16S rRNA Genes from Endophytic Bacteria.

High-throughput sequencing of universal bacterial 16S rRNA gene (16S rDNA) amplicons is a routine method for characterizing bacterial diversity in a range of environments. For eukaryotic host-associated communities, however, plastid and mitochondrial genes are often co-amplified with, and greatly outnumber, bacterial 16S rDNA. This makes it difficult to obtain sufficient numbers of target 16S rDNA sequences to characterize the diversity of endophytic bacterial communities. This chapter describes a method that improves the amplification of bacterial 16S rDNA from plant tissues by using a peptide nucleic acid (PNA) PCR clamp. The PNA clamp selectively binds to a targeted region of the plant genome and inhibits its amplification during PCR. PNA clamps are especially useful for characterizing bacterial communities on plant tissues with lower levels of microbial colonization such as the root tips and leaves.

Detection of Helicobacter pylori in oral yeasts from students of a Chilean university.

Nearly 73% of the Chilean population is infected with Helicobacter pylori (H. pylori), a factor predisposing for gastric cancer. Recent studies have demonstrated the presence of this pathogen within yeasts, suggesting that this fact can directly influence the failure of a treatment, transmission, and reinfection. To detect the presence of H. pylori inside oral yeasts isolated from students of the University of Concepción (Chile). 72 samples, obtained from the oral cavity using cotton swabs were incubated in YPD broth for 48h at 37°C and posteriorly seeded in Sabouraud Dextrose agar plus chloramphenicol at the same temperature and for the same time. Yeasts isolated were observed microscopically (wet mounting and Gram-stained) and identified using microbiological techniques. Intracellular H. pylori detection was performed by the amplification of 16S rDNA by PCR. Oral yeasts were detected in 24 samples (33.3%), being C. albicans (79.2%) the most frequent species, followed by C. dubliniensis (12.4%), C. krusei (4.2%), and C. tropicalis (4.2%). When analyzed by PCR, 15 of the 24 oral yeasts 62.5 % were positive for H. pylori 16S rDNA. From the 15 individuals positive for yeast harboring H. pylori, 81% of them reported stomach discomfort, and the presence of the bacteria was diagnosed at some moment in 20% of them. The intracellular presence of the H. pylori in oral yeasts suggests an endosymbiotic relationship of these microorganisms, which could favor H. pylori transmission and reinfection in the gastrointestinal tract.

Effects of Personal Exposure to Fine Particulate Matter Air Pollutionon Gut microbiotain elderly: The China BAPE study

Background: In recent years, it is confirmed that human gut microbiota is critical for human health. Animal studies suggest that air pollutants may alter the composition of the gut microbiota, which may negatively impact health through changes in the composition or function of the gut microbiome.However, Research on the effects of personal exposure to fine particulate matter on gut microbiota is lacking in developing countries, especially in highly polluted areas.Methods:Participants (60-69 years, n=76) were enrolled between Sep 10th 2018 and Jan 19th 2019 with a month interval.MicroPEMTM real-time personal measurements was used to monitor the subject's personal PM2.5 exposure. Faecal samples from 76 healthy elderly were sequenced using 16S rDNA amplification to assess microbial diversity, richness and relative taxa abundance. Operational Units were clustered with 97% similarity cutoff and chimeric sequences were identified and removed. A linear mixed-effect model was applied to estimate the associations of exposure to Fine Particulate Matter with alpha diversity indices and relative abundance of the gut microbiota. Results:A total of 516 genus level microbiota were identified in this study.The five most abundant bacteria in the fecal flora of the participants were Bacteroides, Prevotella, Pachyphyte, Faecalibacterium and Roseburia.Exposure to fine particulate matter was associated with the alterations in gut microbiota diversity and relative abundance of 20 genus level microbiota. By analyzing the abundance composition of the KEGG pathway of the intestinal flora of the Participants,the pathways enriched by the intestinal flora mainly include transport, DNA repair and recombinant proteins, ribosomes, and purine metabolism. Involves environmental information processing, genetic information processing, and metabolic mechanisms.Conclusions: Exposure to fine particulate matter may alter the relative abundance, and decrease the diversity of the gut microbiota.These findings can extend our understanding of the mechanism of air pollution inducing diseases.

Multicenter analysis of sputum microbiota in tuberculosis patients.

The impact of tuberculosis and of anti-tuberculosis therapy on composition and modification of human lung microbiota has been the object of several investigations. However, no clear outcome has been presented so far and the relationship between M. tuberculosis pulmonary infection and the resident lung microbiota remains vague. In this work we describe the results obtained from a multicenter study of the microbiota of sputum samples from patients with tuberculosis or unrelated lung diseases and healthy donors recruited in Switzerland, Italy and Bangladesh, with the ultimate goal of discovering a microbiota-based biomarker associated with tuberculosis. Bacterial 16S rDNA amplification, high-throughput sequencing and extensive bioinformatic analyses revealed patient-specific flora and high variability in taxon abundance. No common signature could be identified among the individuals enrolled except for minor differences which were not consistent among the different geographical settings. Moreover, anti-tuberculosis therapy did not cause any important variation in microbiota diversity, thus precluding its exploitation as a biomarker for the follow up of tuberculosis patients undergoing treatment.

Phylogenetic characterization and screening of halophilic bacteria from Algerian salt lake for the production of biosurfactant and enzymes

Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).

Ixeris Chinensis is a New Host for Peanut Witches' Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan.

Ixeris chinensis (Thunb. ex Thunb.) Nakai, a perennial herbaceous plant that belongs to the family of Asteraceae, is widely distributed at mid-low altitude regions in Taiwan. I. chinensis is commonly used as traditional herbal medicine for the treatment of inflammation, bronchitis, pneumonia, and diarrhea. In March 2020, disease symptoms such as shoot proliferation, phyllody, virescence, purple top, and witches' broom were observed on I. chinensis at the sansheng community park in Mailiao, Yunlin County, Taiwan. Totally, eight I. chinensis plants were checked and half of them were symptomatic. These disease symptoms are similar to those associated with peanut witches' broom (PnWB) disease identified in the same area (Liu et al. 2015). Three samples mixed with leaf, stem, and flower were tested including one healthy and two symptomatic I. chinensis. The total DNA of each sample was extracted and examined by nested PCR for the amplification of 16S rDNA with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). A specific signal of expected size (1.2 kb) for 16S rDNA was only detected in the symptomatic I. chinensis, but not in healthy I. chinensis. The nucleotide sequence (accession no. MT416114) of the amplified DNA fragment using primer pairs P1/P7 from symptomatic I. chinensis is identical to that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of phytoplasma associated with PnWB disease (Chung et al. 2013). Analysis of the virtual RFLP pattern of MT416114 generated by iPhyClassifier revealed that the phytoplasma detected in symptomatic I. chinensis belongs to a 16SrII-V subgroup. The total protein of each sample was also extracted and examined by western blotting using the polyclonal antibody raised against Imp protein of purple coneflower witches' broom phytoplasma (Chien et al. 2020), which is identical with that (accession no. ADD59806) of PnWB phytoplasma. An expected signal of 19 kDa specific for Imp was detected in symptomatic I. chinensis, but not in healthy I. chinensis. Subsequent PCR, DNA sequencing and western blotting assays further confirmed that the gene encoding a SAP11-like protein was only detected in symptomatic I. chinensis, and shares 100% identity with that (accession no. EMR14684) of PnWB phytoplasma. Our results indicate that PnWB phytoplasma causes disease in I. chinensis, a common weed, which may act as an alternative natural host and facilitate the spreading of phytoplasma disease in Taiwan.

Natural phytoplasma infections on fruit, vegetable and weed plants at the same agroecosystem and their molecular properties

The phytoplasma associated diseases are an emerging threat to fruit and vegetable crops leading severe yield losses worldwide. Pear (Pyrus communis L.) trees, with symptoms of severe reddening, dwarfing and shoot proliferation were observed in pear orchards of Malatya province of Turkey. Tomato (Solanum lycopersicum L.) plants grown nearby the symptomatic pear orchard displaying leaf rolling, severe flower sterility and purple leaves were observed at the same agroecosystem. To verify the presence and diversity of phytoplasmas, symptomatic pears and tomatoes were sampled and weeds nearby the symptomatic plants were collected. Total plant DNA was purified from midrib of collected leaves using a commercial kit. The DNA samples were analyzed by nested polymerase chain reaction (PCR) using universal primer pairs to amplify 16S rDNA fragments. The phytoplasmas detected in collected samples were differed according to the host. Here we detected and characterized ‘Candidatus Phytoplasma pyri’ belonging to apple proliferation group (subgroup 16SrX-C) from a pear tree, ‘Candidatus Phytoplasma trifolii’ belonging to clover proliferation group (subgroup 16SrVI-A) from a weed (Amaranthus retroflexus) and ‘Candidatus Phytoplasma solani’ belonging to the stolbur phytoplasma group (subgroup 16SrXII-A) from a tomato plant. Direct sequencing of PCR products verified the phytoplasmal nature of the infections. The occurrence of ‘Ca. P. trifolii’ on A. retroflexus is the first report for the world. The irregular presence of the phytoplasmas in fruit and vegetable crops and weeds indicates continuous spread of the phytoplasmas threatening the new crops and new horizons.

Optimization of Soil DNA Extraction Protocol Using Na2EDTA, SDS, Heating, Vortexing and CaCl2 and Its Validation for Metagenomic Studies

Aim: Several methods described previously for isolation and purification of soil DNA. Most of these protocols use combination of techniques or methods but the role and contribution of each individual method or component used is not clearly discussed. This study aims at analysing the effect of individual components used in extraction of DNA from soil and finally to optimize soil DNA isolation protocol and its validation by using 16SrDNA sequence analysis.&#x0D; Methods and Results: The soil was washed with anionic buffers before lysis step to reduce humic substances and release microbial cells from soil matrix, then the cells were lysed using combination of SDS, heating and vortexing and finally humic substances were removed using chemical flocculation. Pre-lysis washing of soil with 100 mmol l-1 Na2EDTA proved good for releasing microbial cells from soil matrix. Heating the soil sample at 75°C yielded good quantity (15.73 µg g-1 soil) DNA followed by 2% SDS (10.28 µg g-1 soil) and vortexing at 1400 rpm (8.94 µg g-1 soil). Combination of heating, SDS and vortexing yielded 25 µg DNA per gram of soil. Different concentrations of chemical flocculants like AlNH4(SO4)2, FeCl3, CaCl2 and MgCl2 were used to reduce humic substances. Flocculation with 100 mmol l-1 CaCl2 removed 5.2 mg humic substances without significant loss of DNA. 16S rDNA sequence analysis of DNA extracted from soil reveals presence of all the common soil bacterial species indicating the protocol is unbiased.&#x0D; Conclusion: Combination of chemical (SDS) and physical (heating and vortexing) methods yield good DNA whereas addition of enzyme (lysozyme) did not show significant effect on cell lysis. The digestion of isolated DNA with restriction enzyme and amplification of 16S rDNA using Taq DNA polymerase indicates the isolated DNA is pure enough for metagnomic analysis. 16Sr DNA sequencing of soil DNA indicates that this protocol can extract good quality and quantity DNA from range of bacteria present in soil varying in their cell wall composition. The optimised protocol is unbiased, very simple, does not need special equipments and many samples can be processed simultaneously.

Gut Microbiome Reveals Specific Dysbiosis in Primary Osteoporosis.

Object: Primary osteoporosis (PO) is the most common bone disease, which is characterized by decreased bone mass, damage of bone tissue microstructure, increased bone fragility, and is prone to fracture. Gut microbiome may be involved in bone metabolism of PO through gut–brain axis regulation of immune system and endocrine system, however, the specific mechanism is still unclear. The purpose of this study was to characterize the gut microbiome of patients with PO and its possible role in the occurrence and development of the disease.Methods: Fecal samples were collected from 48 PO patients and 48 healthy controls (HC). The composition of gut microbiome community was analyzed by 16s rDNA amplification sequencing, and the difference of gut microbiome composition between PO patients and HC individuals was compared. PICRUSt was also used to predict the biological function of gut microbiome in patients with PO, and to explore its possible role in the occurrence and development of this disease. The classification model is constructed by random forest algorithm so as to screen the key biomarkers.Result: The diversity of gut microorganisms in PO patients was significantly higher than that in HC group (p < 0.05) and there was significant difference in microbial composition in PO group. The abundance of Dialister (0.036 vs. 0.004, p < 0.001) and Faecalibacterium (0.331 vs. 0.132, p < 0.001) were significantly enriched which were the key flora related to PO. Although no significant correlation between bone mineral density and the richness of microbial communities are found, PICRUST results show that there are a wide range of potential pathways between gut microbiome and PO patients, including genetic information processing, metabolism, environmental information processing, cellular processes, human diseases, and organic systems. Notably, the discriminant model based on dominant microflora can effectively distinguish PO from HC (AUC = 93.56).Conclusions: The findings show that PO is related to the change of gut microbiome, especially the enriched Dialister and Faecalibacterium genera, which give new clues to understand the disease and provide markers for the diagnosis and new strategies for intervention treatment of the disease.